The substrates for cellular functions including transcription, replication, recombination, and chromosome division are chromatin, not naked DNA. These functions are essential for the development and health of all organisms. In the initial level of chromosome organization, DNA is compacted into a repeating array of nucleosomes. The detailed positioning of nucleosomes along DNA can be essential for other negative or positive gene regulation. Distinctive local arrangements of nucleosomes are reported at telomeres and centromeres, in the vicinity of known gene regulatory regions, and at other chromosomal locations; these particular arrangements are believed to be essential for chromosome function. The sequence of the DNA itself strongly biases the position of the nucleosomes into which the DNA is wrapped, and certain DNA sequence rules or motifs involved in nucleosome position have been elucidated. These findings imply that genomic DNA sequences are evolved to facilitate their function through effects on their chromatin structure. The long term aim of this work is to elucidate the relationship between the molecular architecture of chromosomes and their function. The goal of the present project is to explore the relationships between genomic DNA sequence, nucleosome positioning, and chromosome function. May key facts are missing. There is a need for systematic and quantitative analyses, and a need for new experimental tools. We will attack these goals in three ways. These studies build on our recent completion of SLEX experiment which yielded a large number of non-natural DNA sequences have exceptionally high affinity for histone octamer and, consequently, exceptionally strong nucleosome positioning power. (i) We will take similar approaches to select from the entire yeast genome those regions have the greatest power for DNA sequence-directed nucleosome positioning. These sequences will be characterized, their free energy for nucleosome positioning quantified, and their contributions to chromosome function analyzed. (ii) We will use our existing selected positioning sequences to manipulate and study nucleosome positioning in vivo and we will use biophysical approaches in vitro to analyze the DNA structural and mechanical properties that are responsible for their strong positioning power. (iii) We will take both evolutionary and design approaches to create a next generation of even stronger positioning sequences for use in future studies in vitro and in vivo.